The present invention relates generally to the use of a light absorbing wall material to eliminate stray light paths in light-guiding applications, such as High Performance Liquid Chromatography (HPLC), and Capillary Zone Electrophoresis (CZE) spectroscopic analysis.
Systems for light absorption detection generally comprise four basic components; a light source, a means for selecting wavelengths to be used, a light-guiding vessel, typically in the form of a hollow tube or capillary through which a sample to be analyzed and light are passed (a flowcell), and a light detector which measures the amount of light transmitted through the flowcell. Large optical throughput can be achieved when the light is guided along the capillary similar to the way light is guided along an optical fiber.
A flowcell must be constructed from materials that are resistant to the solutions encountered in liquid chromatography or CE. To achieve high sensitivity to small concentrations of analyte, the cell must have a high optical throughput and a long pathlength. If the quantity of analyte is small and capillary separation techniques are used, the volume of the cell must also be small, otherwise band spreading and loss of chromatographic resolution occurs. The transmittance, T, of light through such a system filled with a light absorbing sample is determined in accordance with Beer's law:
                    T        =                  I                      I            0                                              (                  1          ⁢          a                )                                A        =                                            log              10                        ⁡                          (                                                I                  0                                I                            )                                =                      ɛ            ⁢                                                  ⁢            bc                                              (                  1          ⁢          b                )            
where I0 is the light exiting the flowcell when it is filled with clear mobile phase and I is the light power exiting the flowcell when analyte is present. b is the path length of the flowcell conventionally expressed in centimeters, c is the analyte concentration in M or moles/liter and ε is the molar absorptivity expressed in units of cm−1(moles/liter)−1. A is the absorbance, a dimensionless number expressed in absorbance units (au).
The requirement for high light throughput and long pathlength is illustrated by differentiating equation (1b).
                              Δ          ⁢                                          ⁢          c                =                              Δ            ⁢                                                  ⁢            A                                b            ⁢                                                  ⁢            ɛ                                              (                  1          ⁢          c                )            
Δc represents the smallest analyte concentration that can be detected and ΔA the corresponding smallest change in absorbance that can be measured. This represents the noise at the absorbance baseline, the output of the absorbance detector.
As illustrated by equation (1b), low absorbance noise requires a high light signal I0 and low noise in the measurement of I, i.e. a high signal-to-noise (S/N) ratio in the raw transmittance measurement. In a well-designed detector, shot noise, which is proportional to the square root of the light signal, dominates, so high S/N requires high light throughput.
Light-guiding flowcells enable low volume cells to be constructed with high light throughput and long path length. The liquid sample is contained in a tube of material having a lower refractive index (RI) than the mobile phase. Light is introduced into one end of the tube and propagates down the axis of the tube making multiple internal reflections before emerging at the other end. The liquid is analogous to the core of an optical fiber and the material of the tube is analogous to the cladding. The condition for light guiding is that the rays incident on the liquid/wall boundary do so at an angle of incidence greater than the critical angle θc.
                              θ          c                =                              sin                          -              1                                ⁢                                    n              2                                      n              1                                                          (                  2          ⁢          a                )            where n1 is the RI of the liquid, and n2 is the RI of the wall of the flowcell.
The numerical aperture (NA) of the guided beam is given by:NA=sin−1φ=(n12−n22)1/2  (2b)Where φ is the largest angle, between a ray entering the cell from air and the cell axis, which meets the guiding condition. The guiding mechanism is termed total internal reflection (TIR)
Recently, flowcells having an inner surface of an amorphous fluoropolymer material that has an index of refraction lower than that of common chromatography solvents, e.g. water, have enabled light-guiding flowcells to be constructed.
Light introduced along the axis of the tube is guided in the fluid by total internal reflection at the fluid wall boundary. The amorphous fluoropolymer material Teflon® AF 1600 and 2400 are preferred tube materials because they are transparent throughout the visible and ultraviolet spectrum, they have an unusually low refractive index (1.31 and 1.29 respectively) and are chemically inert. As a comparison, the RI of water at the same wavelength is 1.333. All common solvents (as methanol/water mixtures and acetonitrile) have a higher RI than water and therefore also Teflon® AF. Only pure methanol has an index slightly below water, but still above that of Teflon® AF. Even at different wavelengths, the fluoropolymers retain the RI advantage.
However, it is difficult to construct a cell with amorphous fluoropolymer walls without some light entering the end cross-section of the wall, or some light 18 being scattered into the wall from the liquid, or some light 18 entering the fluid from the walls after bypassing part or all of the sample fluid. These aberrant light paths result in a stray light background and inaccuracy in the readings, limiting the linearity and dynamic range of a detector that is supposed to receive only light that has passed through the liquid.
One strategy to control stray light positions opaque masks between the walls and the light, but the small diameter tubes of HPLC and CZE equipment makes the alignment of such masks difficult and time consuming. A second strategy to control stray light supplies entering light through an optical fiber having an OD that fits between the walls, but the amount of light coupled into the liquid is reduced geometrically by the reduced area
The difficulty of controlling stray light becomes greater as fluid cross-sections are made smaller. For capillary HPLC or CZE detection, a fluid channel ID of 100 μm or less is needed to create a small volume flowcell, with sufficient pathlength to preserve analytical sensitivity. A better way is needed to fabricate light guiding flowcells to avoid the difficulties of controlling stray light outlined above